Method of expressing recombinant protein in cho cells

ABSTRACT

Method of expressing recombinant protein in CHO cells, by using an expression vector comprising the murine IgG 2A gene locus.

The present invention relates to a method for expressing a recombinantproduct gene in a CHO cell line as well as to recombinant CHO host cellsand to novel expression vector constructs.

The Chinese Hamster ovary cell (CHO) mammalian expression system iswidely used in production of recombinant protein. Apart from lymphoidcell lines such as hybridoma cell lines, it is one of the few cell typesallowing for simple and efficient high-density suspension batch cultureof animal cell. Furthermore, they allow for very high product yields andare comparatively robust to metabolic stresses whereas lymphoid cellsare more difficult to culture at an industrial scale. Given considerablecost of production, it is of utmost importance to maximize the yield ofrecombinant protein per bioreactor run. Choice of culture mediumcomposition and bioreactor design and operation are parameters thatimpact yield and may be quite complex to optimize. More predictably,increases in the strength or transcriptional activity of the promotercontrolling expression of product protein enhance yield. Incrementalincreases at the single cell level will translate into considerableimprovements of product yield in high-density batch or fed-batch cultureshowing stationary phase gene expression at cell densities in the rangeof 10⁶ to 10⁷ cells/ml.

U.S. Pat. No. 5,866,359 describes a method of enhancing expression froman already strong hCMV promoter in CHO and NSO cells by co-expressiongadenoviral E1A protein from a weak promoter. E1A is a multifunctionaltranscription factor which may act on cell cycle regulation and has bothindependent transcriptional activating and repressing functionaldomains. The finetuning of E1A expression to appropriate low levelexpression is crucial for success of the co-expression approach in orderto achieve the ideal balance in between gene transactivation whilstavoiding any negative impact on cell cycle progression. As adisadvantage, apart from careful choice of the promoter driving E1Aexpression, this system blocks part of the protein synthesis capacity ofthe cell with E1A expression rather than expressing the recombinantprotein of interest.

WO 95/17516 describes use of the murine immunoglobulin gamma 2A locusfor targetting an expression vector construct to a highly active genelocus in lymphoid cells of the B-cell lineage, e.g. widely used NSOmyeloma cells. NSO cells essentially are a tumor cell line of murineplasma or B-cells. Only in B-cells, the chromatin harboring theimmunoglobulin loci is in its fully active, open state, allowing forhigh transcriptional activity of native immunoglobulin promoters orrecombinant expression constructs integrated into those gene loci.

As a disadvantage, due to the principle of homologous recombination, thetargetting sequence will target efficiently in murine cell lines onlymatching the sequence of the gamma 2A targetting sequence harboring arecombinatorial hot spot; for high level expression, the gamma 2A locusregion must be a transcriptionally active genomic region, limiting itseffectiveness for homologous recombination to B-cell types.

It is an object of the present invention to devise another expressionsystem for CHO protein expression in biotechnology which allows forenhanced expression from a standard promoter. According to the presentinvention, this aim is surprisingly achieved by equipping a geneexpression vector for CHO cells with a gene targetting sequence havingbeen originally devised for homologous recombination in murine B-cells.

Possible embodiments of the invention are shown in the figures. What isshown is:

FIG. 1 Relative expression levels of green fluorescent protein (GFP)from hCMV promoter and hCMV promoter in the presence of the IgG 2A hotspot sequence in transient transfection of CHO-K1 cells

FIG. 2 Relative GFP expression levels from hCMV promoter and hCMVpromoter in the presence of the IgG 2A hot spot sequence in stablytransfected CHO-K1 cells.

FIG. 3 Plasmid map of hCMV-MIE expression vector carrying IgG 2Atargetting sequence

According to the present invention, a DNA sequence for expression of arecombinant gene in a mammalian cell comprises a recombinant productgene and a promoter for expressing the recombinant product gene,preferably a CMV promoter, and further comprises a murine immunoglobulingamma 2A locus DNA sequence or fragments or sequence variants thereofcapable of enhancing expression from the promoter. According to thepresent invention, such a DNA sequence is useful expression vectorconstruct for expression of recombinant product gene in CHO cells.

According to the present invention, the method of expressing arecombinant protein comprises the steps of

-   -   a. culturing a CHO cell transfected with an expression vector        comprising a promoter active in CHO cells driving expression of        a recombinant product protein and further comprising the murine        IgG 2A gene locus DNA or a DNA sequence variant or DNA fragment        thereof which is enhancing activity of said promoter, and    -   b. harvesting the product protein

A recombinant product gene according to the present invention is theproduct protein that is sought to be expressed and harvested in highamount. It may be any protein of interest, e.g. therapeutic proteinssuch as interleukins or enzymes or subunits of multimeric proteins suchas antibodies or fragments thereof. The recombinant product gene mayinclude a signal sequence coding sequence portion allowing secretion ofthe once expressed polypeptide from the host producer cell. In a furtherprefered embodiment of the present invention, the product protein is asecreted protein. More preferably, the first or product protein is anantibody or engineered antibody or a fragment thereof, most preferablyit is an Immunoglobulin G (IgG) antibody.

The DNA sequence of the murine immunoglobulin gamma 2A gene locus (IgG2A) has originally been devised in WO 95/17516 for use as a genomictargetting sequence for generating stably recombinant lymphoid B-celllines that show high expression of the recombinant gene product. Blymphocytes or plasma cells normally express extremely high levels ofimmunoglobulin RNA from the the Ig heavy chain locus, probably due tocell-type specific enhancer/transcription factor activity and openchromatin structure. The preferred murine immunoglobuline gamma 2A genesequence of the present invention is the same as the targetting sequenceused in WO 95/17516. It is a 5.1 kb BamHI genomic fragment whichincludes all of the coding region of murine Ig gamma 2A except the most5′ part of the CH1 exon (Yamawaki-Kataoka, Y. et al., Proc. Natl. Acad.Sci. U.S.A. (1982) 79: 2623-2627; Hall, B. et al., Molecular Immunology(1989) 26:819-826; Yamawaki-Kataoka, Y. et al., Nucleic Acid Research(1981) 9: 1365-1381). According to the present invention, promotion ofsite-directed, homologous recombination is not the relevant property ofthe immunoglobulin gamma 2A gene sequence (IgG 2A).

Accordingly, any sequence variant of said IgG 2A gene sequence orsequence fragment or variant sequence fragment that is functional in orcapable of enhancing recombinant product gene expression from thepromoter, preferably from a hCMV promoter as set forth below, both undercondition of transient or stable expression in CHO cells is alsoencompassed by the present invention.

Such ‘functional’ variants encompass e.g. base insertions, deletions orpoint mutations and be generated by methods well-known in the art, e.g.by primer-directed PCR, ‘error-prone’ PCR, ‘gene-shuffling’ termedPCR-reassembly of overlapping DNA fragments or by in-vivo randommutagenesis of bacterial clones followed by library transfection andfunctional selection in CHO cells. For instance, random mutagenesis canbe achieved by alkylating chemicals or UV-irradiations as described inMiller, J., Experiments in Molecular Genetics, Cold Spring HarborLaboratory 1972). Optionally, a natural mutator-strain of a hostbacterium may be used.

Preferably, such variant sequence or sequence fragment is at least 65%,more preferably 75%, most preferably 90% homologous in DNA sequence tothe corresponding part of the natural murine immunoglobuline gamma 2Agene locus. For instance, it is possible to insert a Sal I restrictionsite at the naturally occurring Stu I site present 39 bp upstream ofmembrane exon 2 (M2) to provide a unique site for linearization withinthe murine immunoglobulin gamma 2A sequence; such sequence variant wasoriginally devised for site-specific recombination targetting, but canas well be employed in the context of the present invention.

A ‘promoter’ is defined as a DNA sequence that directs RNA polymerase tobind to DNA and intiates RNA synthesis. According to the presentinvention, it is a promoter that is active in CHO cells. Such a promoterpreferably is a strong promoter. A strong promoter is one which causesmRNAs to be initated at high frequency equal to or higher than that ofhCMV core promoter/enhancer fragment (described in U.S. Pat. No.5,168,062) in CHO-KI cells. Such promoter may be a cell-type dependentstrong promoter, as are cited in U.S. Pat. No. 5,589,392, or preferablyis a ubiquitously active strong promoter, more preferably aconstitutively active viral promoter such as e.g. early and latepromoters of the SV40 virus, the immediate early promoter of the humancytomegalovirus (hCMV) or of murine cytomegalovirus (mCMV), thethymidine kinase promoter (TK) of Herpes Simplex virus or the RousSarcoma Virus long terminal repeat promoter (RS-LTR), more preferably itis the hCMV-MIE promoter as defined by the 2.1 kb Pst I fragmentdescribed in U.S. Pat. No. 5,385,839 and/or EP-323 997-A1 or afunctional part thereof having promoter activity. The hCMV promoterconstruct harboring the complete first functional intron of the majorimmediate early (MIE) gene of hCMV, as set forth in EP-323 997-A1, is aparticularly preferred embodiment of the present invention.

Preferably a hCMV promoter employed in the present invention lacks the‘modulator’ sequence part in the upstream/enhancer portion of thepromoter. The ‘modulator’ sequence has been found to be detrimental tohCMV promoter activity in CHO cells and stretches from position −750 toposition −1150 relative to the MIE transcription start site (Meier etal., 1996, Intervirology 39: 331-342, Regulation of hCMV immediate-earlygene expression), in particular in transient transfection. Without themodulator sequence, the enhancing effect of the presence of the IgG 2Ahost spot sequence on (modulator negative or mod- for short) hCMVpromoter is even more pronounced.

A transient transfection is characterised by non-appliance of anyselection pressure for a vector borne selection marker. A pool or batchof cells originating from a transient transfection is a pooled cellpopulation that comprises cells which have taken up and do express andcells that have not taken up the foreign DNA. Cells that express theforeign expression cassette do usually not have integrated thetransfected DNA into their genome yet and tend to lose the foreign DNAand to overgrow transfected cells in the population upon culture of thetransiently transfected cell pool. Therefore expression is strongest inthe period immediately following transfection and decreases with time.Preferably, a transient transfectant according to the present inventionis understood as a cell that is maintained in cell culture in theabsence of selection pressure up to a time of 90 hours posttransfection.

Preferably, a transfected CHO host cell according to the presentinvention is a stably transfected host cell, in particular incombination with a hCMV promoter as set forth above. Stable transfectionmeans that newly introduced foreign DNA is becoming incorporated intogenomic DNA, usually by random, non-homologous recombination events; incase of a vector sequence, stable transfection according to the presentinvention may result in loss of vector sequence parts not directlyrelated to expression of the recombinant product gene, such as e.g.bacterial copy number control regions rendered superfluous upon genomicintegration. A transfected host cell has integrated at least part ordifferent parts of the expression vector into the genome. Likewise,transfection of CHO cells with two or several DNA fragments giving riseat least in vivo to functional equivalents of the essential elements ofthe expression vector of the invention, namely the product gene undercontrol of a suitable promoter and the hot spot IgG 2A sequence, iscontained in the definition of such transfected host cells. In vivoassembly of functional DNA sequences after transfection of fragmentedDNA is described e.g. in WO 99/53046. It is possible that such stableintegration gives rise, upon exposure to further selection pressure forgene amplification, to double minute chromosomes in CHO cells. This iscomprised in the present meaning of ‘stable’. Upon random genomicintegration of the expression vector of the present invention in CHO,the presence of the targetting sequence enhances promoter activity forexpression of the recombinant product protein. Such effect has not beenobserved nor could it have been anticipated upon homologous genetargetting in mature murine B-cell lines including plasmacytoma/myelomacell lines; there, the IgG 2A targetting sequence served solely toincrease the frequency of high-yielding homologous integrants since theIgG 2A locus proved to be a recombinatorial ‘hot spot’. As said before,the chromatin of the immunglobuline genomic region is in an open, highlyactive state in suitably targetted B-cell lines.

‘Expression vectors’ are defined herein as DNA sequences that arerequired for transcription and the translation of their mRNAs in anappropriate mammalian host cell line after transfection with vector. Anappropriately constructed expression vector should usually contain: atleast one expressable marker selectable in animal cells, a limitednumber of useful restriction sites for insertion of the expressioncassette for the recombinant product gene under control of an upstreampromoter region. Where used in particular for transient/episomalexpression only, it may further comprise an origin of replication suchas origin of Eppstein Barr Virus (EBV) or SV40 virus for autonomousreplication/episomal maintenance in eukaryotic host cells but may bedevoid of a selectable marker. Expression vectors are e.g., but are notlimited to, linear DNA fragments, DNA fragments encompassing nucleartargeting sequences or are specially optimized for interaction withtransfection reagents, animal viruses or suitable plasmids that can beshuttled and produced in bacteria. Any selection marker commonlyemployed such as thymidine kinase (tk), dihydrofolate reductase (DHFR)or glutamine synthetase (GS) may be used. In a preferred embodiment, anexpressable GS selection marker is employed (Bebbington et al., 1992,High-level expression of a recombinant antibody from myeloma cells usinga glutamine synthetase gene as an amplifiable selectable marker,Bio/Technology 10:169-175; Cockett et al., 1990, High level expressionof tissue inhibitor of metalloproteinases in Chinese Hamster Ovary (CHO)cells using Glutamine synthetase gene amplification, Bio/Technology 8:662-667).—The GS-system is one of only two systems that are ofparticular importance for the production of therapeutic proteins. Incomparison to the dihydrofolate reductase (DHFR) system, the GS systemoffers a large time advantage during development because highlyproductive cell lines can often be created from the initial tranfectantthus avoiding the need for multiple rounds of selection in the presenceof increasing concentrations of selective agent in order to achieve geneamplification (Brown et al., 1992, Process development for theproduction of recombinant antibodies using the glutamine synthetase (GS)system, Cytotechnology 9:231-236). It goes without saying thatequivalent to a second transcription unit for expression of the markergene, an expression unit could use a monocistronic expression cassetteboth for the product gene and the marker gene by employing e.g. internalribosome entry sites as is routinely employed in the art. Vice versa, itgoes without saying that the hot spot IgG 2A sequence of the presentinvention and the expression cassette for the product protein comprisinga promoter and/or marker cassette are not required to work in cis on asingle expression vector; the elements can be well carried on separateco-transfected vectors or DNA fragments which may then be chromosomallyintegrated at a single, concatemeric integration site.

A further object of the present invention are CHO host cells transfectedwith the DNA sequences of the present invention. Further objects are amethod for transfection of such host cells and a method for expressionof the recombinant product gene in such host cells. The explanations andreferences made to preferred embodiments in the present specification ofthe invention relate likewise to all these further objects of thepresent invention. It is to be noted that a host cell transfected withthe DNA sequence or vector of the present invention is to be construedas being a transiently or stably transfected cell line. Any transfectiontechnique such as those well-known in the art, e.g. electoporation,Ca-phosphate precipitation, DEAE-dextrane transfection, lipofection, canbe employed according to the present invention if appropriate for agiven host cell type.

A suitable host cell line can be any chinese hamster ovary (CHO) cellline (Puck et al., 1958, J. Exp. Med. 108: 945-955). The term ‘hostcell’ refers to cells capable of growth in culture and expressing adesired protein recombinant product protein. Suitable cell lines can bee.g. CHO K1 (ATCC CCL-61), CHO pro3-, CHO DG44, CHO P12 or the dhfr-CHOcell line DUK-BII (Chassin et al., PNAS 77, 1980, 4216-4220) or DUXB11(Simonsen et al., PNAS 80, 1983, 2495-2499). In CHO cells, theimmunoglobuline gene loci are inactive and the chromatin is therefore ina densely packaged or closed state. Thus, any gene construct integratedin the immunoglobuline loci could not give rise to high-level expressionof recombinant protein due to the specific state of chromatin, unless itwould itself comprise flanking locus control regions promoting openingof the chromatin on both sides of the expression cassette. Further,immunoglobuline gene sequence, and in particular the intron portions ofit, show considerably divergence amongst species, e.g. from mouse tohamster. The promoter or enhancer elements of immunoglobline loci arebothspecies and tissue specific and should be active in B-cells only.The murine IgG 2A sequence of the present invention enhances geneexpression in CHO cells also in the absence of any naturalimmunoglobuline promoter that is giving rise to full-length transcriptscoding for complete IgG heavy chain. Preferably, the IgG 2A sequence ofthe present invention is devoid of such promoter. Surprisingly, themurine IgG 2A targetting sequence even improved gene expression in CHOcells upon transient transfection of CHO cells with expression vectorsaccording to the present invention (FIG. 1); such transient expressionis a further preferred embodiment of a method according to the presentinvention. In transient expression assays which are commonly takingplace about 20-50 hours post transfection, the transfected vectors aremaintained as episomal elements and are not yet integrated into thegenome. Suitable media and culture methods for mammalian cell lines arewell-known in the art, as described in U.S. Pat. No. 5,633,162 forinstance. Examples of standard cell culture media for laboratory flaskor low density cell culture and being adapted to the needs of particularcell types are for instance: Roswell Park Memorial Institute (RPMI) 1640medium (Morre, G., The Journal of the American Medical Association, 199,p. 519 f. 1967), L-15 medium (Leibovitz, A. et al., Amer. J. of Hygiene,78, 1p. 173 ff, 1963), Dulbecco's modified Eagle's medium (DMEM),Eagle's minimal essential medium (MEM), Ham's F12 medium (Ham, R. etal., Proc. Natl. Acad. Sc. 53, p 288 ff. 1965) or Iscoves' modified DMEMlacking albumin, transferrin and lecithin (Iscoves et al., J. Exp. med.1, p. 923 ff., 1978). For instance, Ham's F10 or F12 media werespecially designed for CHO cell culture. Other media specially adaptedto CHO cell culture are described in EP-481 791. It is known that suchculture media can be supplemented with fetal bovine serum (FBS, alsocalled fetal calf serum FCS), the latter providing a natural source of aplethora of hormones and growth factors. The cell culture of mammaliancells is nowadays a routine operation well-described in scientifictextbooks and manuals, it is covered in detail e.g. in R. Ian Fresney,Culture of Animal cells, a manual, 4th edition, Wiley-Liss/N.Y., 2000.

Preferably, the cell culture medium according to the present inventionis devoid of fetal calf serum (FCS or FBS), which then is being termed‘serum-free’. Cells in serum-free medium generally require insulin andtransferrin in a serum-free medium for optimal growth. Transferrin mayat least partially be substituted by non-peptide chelating agents orsiderophores such as tropolone as described in WO 94/02592 or increasedlevels of a source of anorganic iron favorably in conjunction withantioxidants such as vitamin C. Most cell lines require one or more ofsynthetic growth factors (comprising recombinant polypeptides),including e.g. epidermal growth factor (EGF), fibroblast growth factor(FGF), insulin like growth factors I and II (IGFI, IGFII), etc. Otherclasses of factors which may be necessary include: prostaglandins,transport and binding proteins (e.g. ceruloplasmin, high and low densitylipoproteins, bovine serum albumin (BSA)), hormones, includingsteroid-hormones, and fatty acids. Polypeptide factor testing is bestdone in a stepwise fashion testing new polypeptide factors in thepresence of those found to be growth stimulatory. Those growth factorsare synthetic or recombinant. There a several methodological approacheswell-known in animal cell culture, an exemplary being described in thefollowing. The initial step is to obtain conditions where the cells willsurvive and/or grow slowly for 3-6 days after transfer fromserum-supplemented culture medium. In most cell types, this is at leastin part a function of inoculum density. Once the optimal hormone/growthfactor/polypeptide supplement is found, the inoculum density requiredfor survival will decrease. In a more preferred embodiment, the cellculture medium is protein-free, that is free both of fetal serum andindividual protein growth factor supplements or other protein such asrecombinant transferrin.

A possible embodiment of one method of the present invention, namelyexpression and harvest of the recombinant product protein, ishigh-density growth of the animal host cells e.g. in an industrialfed-batch bioreactor. Conventional downstream processing may then beapplied. Consequently, a high-density growth culture medium has to beemployed. Such high-density growth media can usually be supplementedwith nutrients such as all amino acids, energy sources such as glucosein the range given above, inorganic salts, vitamins, trace elements(defined as inorganic compounds usually present at final concentrationsin the micromolar range), buffers, the four nucleosides or theircorresponding nucleotides, antioxidants such as Glutathione (reduced),Vitamine C and other components such as important membrane lipids, e.g.cholesterol or phosphatidylcholine or lipid precursors, e.g. choline orinositol. A high-density medium will be enriched in most or all of thesecompounds, and will, except for the inorganic salts based on which theosmolarity of the essentially isotonic medium is regulated, comprisethem in higher amounts (fortified) than the afore mentioned standardmedia as can be incurred from GB2251 249 in comparison with RPMI 1640.Preferably, a high-density culture medium according to the presentinvention is balancedly fortified in that all amino acids except forTryptophane are in excess of 75 mg/l culture medium. Preferably, inconjunction with the general amino acid requirement, Glutamine and/orAsparagine are in excess of 1 g/l, more preferably of 2 g/l ofhigh-density culture medium. In the context of the present invention,high-density cell culture is defined as a population of animal cellshaving temporarily a density of viable cells of at least or in excess of10⁵ cells/ml, preferably of at least or in excess of 10⁶ cells/ml, andwhich population has been continously grown from a single cell orinoculum of lower viable cell density in a cell culture medium in aconstant or increasing culture volume.

In a further prefered embodiment, the fed-batch culture is a culturesystem wherein at least Glutamine, optionally with one or several otheramino acids, preferably glycine, is fed to the cell culture as describedin GB2251249 for maintaing their concentration in the medium, apart fromcontrolling glucose concentration by spearate feed. More preferably, thefeed of glutamine and optionally one or several other amino acids iscombined with feeding one or more energy sources such as glucose to thecell culture as described in EP-229 809-A. Feed is usually initiated at25-60 hours after start of the culture; for instance, it is useful tostart feed when cells have reached a density of about 10⁶ cells/ml. Itis well known in the art that in cultured animal cells, ‘glutaminolysis’(McKeehan et al., 1984, Glutaminolysis in animal cells,in: CarbohydrateMetabolism in Cultured Cells, ed. M. J. Morgan, Plenum Press, New York,pp. 11-150) may become an important source of energy during growthphase. The total glutamine and/or asparagine feed (for substitution ofglutamine by asparagine, see Kurano, N. et al., 1990, J. Biotechnology15, 113-128) is usually in the range from 0.5 to 10 g per l, preferablyfrom 1 to 2 g per l culture volume; other amino acids that can bepresent in the feed are from 10 to 300 mg total feed per litre ofculture, in particular glycine, lysine, arginine, valine, isoleucine andleucine are usually fed at higher amounts of at least 150 to 200 mg ascompared to the other amino acids. The feed can be added asshot-addition or as contionusly pumped feed, preferably the feed isalmost continously pumped into the bioreactor. It goes without sayingthat the pH is carefully controlled during fed-batch cultivation in abioreactor at an approximately physiological pH optimal for a given cellline by addition of base or buffer. When glucose is used as an energysource the total glucose feed is usually from 1 to 10, preferably from 3to 6 grams per litre of the culture. Apart from inclusion of aminoacids, the feed preferably comprises a low amount of choline in therange of 5 to 20 mg per litre of culture. More preferably, such feed ofcholine is combined with supplementation of ethanolamine essentially asdescribed in U.S. Pat. No. 6,048,728, in particular in combination withfeeding glutamine. It goes without saying that upon use of the GS-markersystem, lower amounts of glutamine will be required as compared to anon-GS expression system since accumulation of excessive glutamine inaddition to the endogenously produced would give rise to ammoniaproduction and concomittant toxicity. For GS, glutamine in the medium orfeed is mostly substituted by its equivalents and/or precursors, that isasparagine and/or glutamate.

It is a further, independent object of the present invention to devisean expression vector comprising at least a (first) transcription unitfor a product gene, giving rise to product protein upon expression in ahost cell, and which transcription unit is under the control of themouse Cytomegalovirus promoter (mCMV promoter), and further comprising asecond transcription unit comprising a glutamine synthetase (GS) markergene. Such a product gene, or gene of interest (GOI) as it may betermed, can be e.g. an immunoglobulin coding sequence. A glutaminesynthetase marker gene is any enzymatically active GS coding sequence,be it a natural gene sequence or a variant thereof. The abovedefinitions of ‘functional variant’ as set forth above apply here aswell including the preferred ranges of sequence homology. Preferably,the GS marker gene is a mammalian GS marker gene or derived thereof.Surprisingly, such expression vector allows for much higher transfectionrates upon transfection in CHO cells than does e.g. an expression vectorin which the first transcription unit harboring the gene of interest isunder control of the hCMV promoter. This despite the fact that in CHOcells, transcriptional activity of the mCMV promoter is much higher thanthat of hCMV promoter; usually it is believed that upon transfection,higher metabolic load reduces clonal survival upon transfection,resulting in lower numbers of transfectants. Thus the effect can not becorrelated in an obvious manner with the amount or unexpected toxicityof product protein expressed, the latter possibly adversely affectinggrowth of transfectants. Indeed, the finding is the very opposite of anyexpectation of a skilled person.

Further objects according to the present invention are animal hostcells, in particular CHO cells, transfected with such an expressionvector which vector can be maintained episomally or can be stablyintegrated in the genome and a respective transfection method. Likewise,transfection of animal cells, in particular CHO cells, with two or moregene fragments giving rise in-vivo to functional equivalents of thetranscription units of the present object of the invention, is withinthe definition of such transfected host cells. Preferably, said hostcells are stably transfected cells, meaning that the first and secondtranscription unit are chromosomally integrated.

A further object is the use of mCMV promoter to enhance transfectionrate in CHO cells, preferably when using an expression vector comprisingat least a first transcription unit for a product gene which first unitis giving rise to product protein upon expression in a host cell andwhich first transcription unit is further under the control of the mouseCytomegalovirus promoter (mCMV promoter), and further comprising asecond transcription unit comprising a glutamine synthetase (GS) markergene. It may also be possible to transfect the first and secondexpression borne on different vectors, or as isolated gene fragmentsharboring individual expression units. Further, it may be possible totransfect a CHO cell that is already recombinant for and expresses GSwith a first transcription unit harboring mCMV. According to the presentinvention, ‘enhancing transfection rate’ is defining by comparingtransfection rate in the presence of the mCMV promoter and expressionvector according to the present invention with the transfection rate ofthe same expression vector and host cell under identical transfectionand cell culture conditions except that in the expression vector, themCMV promoter is substituted to the hCMV-first intron enhancer/promoterconstruct as defined in U.S. Pat. No. 5,658,759 and as set forth e.g. insequence ID. No. 3 of the present invention. This hCMV-intronMIE-promoter construct, for a given identical product gene, serves as astandard for determining the claimed effect of enhanced transfectionrates. Preferably, use of mCMV promoter results in at least 10-timesenhanced transfection rate.

All relevant definitions given further above apply likewise to thepresent, independent objects of the invention. It must be stressed thatthe present object of the invention does not require the presence of themurine IgG 2A targetting sequence as a prerequisite.

Murine cytomegalovirus (mCMV) is a member of the highly diverse group ofherpesviridae. Even amongst cytomegaloviruses of different host speciesthere can be wide variation. For example, mCMV differs considerably fromthe human cytomegalovirus (hCMV) with respect to biological properties,immediate early (IE) gene organization, and overall nucleotide sequence.The 235-kbp genome of mCMV also lacks large internal and terminal repeatcharacteristics of hCMV. Accordingly, no isomeric forms of the MCMVgenome exist (Ebeling, A. et al., (1983), J. Virol. 47, 421-433; Mercer,J. A. et al., (1983), Virology 129, 94-106). According to the presentinvention, it is possible to employ the promoter region essentiallycorresponding to a large approx. 2.1 kb PstI fragment described in U.S.Pat. No. 4,968,615 or any functional fragment thereof. In a morepreferred embodiment, the mCMV promoter fragment employed comprises thetranscription start site (+0) and extends upstream to about position−500. Surprisingly, such fragment has been found to promote strongerexpression than a promoter cassette extending 800 bp further upstreambeyond position −500. In a most preferred embodiment, a core promoterregion is employed that extends from the transcription start siteupstream but to the Xho I restriction site at about position −150 fromthe natural transcription start site or even extending but to position−100 upstream from the natural transcriptions start site. It goeswithout saying that the transcription start site might be engineered inorder to comprise a suitable restriction site for insertion of therecombinant product gene.

According to the present invention, it is also possible that the firsttranscription unit that is under control of the mCMV promoter harbors atleast one intron sequence. Such measure is well-known in the art forstabilising RNA transcripts and for promoting efficient proteinsynthesis from the corresponding MRNA. For efficient protein synthesiswithout having regard to the claimed effect on transfection rate, it ishowever not advisable to include the first, natural intron of mCMV inthe mCMV promoter construct. In contrast to the situation with hCMVpromoter (cf. U.S. Pat. No. 5,591,639), such natural first intron ofmCMV was found to decrease expression of a recombinant gene from themCMV promoter and is therefore excluded in a further preferredembodiment.

Examples of preferred, possible embodiments of GS marker gene cassettesare given in the sequence listings. Seq IDs No. 1 (pEE 15.1 hCMV/GFP+hotspot)+2 (pEE 14.4 hCMV/GFP) give examples of suitable GS-gene cassettesthat are expressed from the SV40 (early and late, respectively)promoter, a weak to medium level promoter, further comprising anexpression cassette for GFP (Green fluorescent protein) that is undercontrol of the hCMV promoter. Seq. ID No. 1 describes a GS cDNA sequencedescribed in more detail in the figure legend of FIG. 3, under controlof the SV40 early promoter. Seq. ID No. 2 specifies an artificalGS-minigene cassette comprising an intron that is under control of theSV40 late promoter. CHO cells are not naturally glutamine auxothropic,therefore selection schemes as e.g. described in Cockett et al., 1990,High level expression of tissue inhibitor of metalloproteinases inChinese Hamster Ovary (CHO) cells using Glutamine synthetase geneamplification, Bio/Technology 8: 662-667, can be applied. Examples ofsuitable transfection methods for CHO cells are equally given therein;it is possible to employ e.g. classic calcium phosphate precipitation ormore modern lipofection techniques. Transfection rate is routinelydefined as the number of positively transfected cells (transienttransfection) or clones (stable transfection after selection period)obtained from a pool of cells subjected to transfection. The purportedeffect of the present object of invention can be seen e.g. bytransfecting CHO-K1 cells by lipofection (any commercial s reagent andmanufacturers protocol) with the plasmids of either Seq. ID No. 3 (pEE12.4 hCMV-GFP+SV40 early promoter/GS cDNA) or Seq. ID No. 4 (pEE 12.4mCMV-GFP +SV40 early promoter/GS cDNA). Transfected cells may be grownin any conventional culture medium. The culture medium may be a fetalserum-supplemented or serum-free medium as has been defined above.Preferably, the cell culture medium is a serum-supplemented medium, morepreferably a cell culture medium that has been supplemented with atleast 1% (v/v) fetal serum, most preferably with at least 5% (v/v) fetalserum such as fetal calf serum or fetal bovine serum. In anotherpreferred embodiment, the transfection method carried out iselectroporation.

EXPERIMENTS Experiment 1

Transient and Stable Expression of GFP Vector Comprising Hot SpotSequence in CHO-K1 Cells

CHO-K1 cells (ATCC CCL-61) were adapted and cultured in normal cellculture medium GMEM-S (Gibco, UK) with 10% FCS.—For GS selection, themedium must be completely free of glutamine as set forth in table 1below; this necessitates use of dialysed FCS.—All culturing was carriedout in shake flask at 36.5° C. with orbital shaking at 125 rpm.Lipofectin (Superfectin™,Gibco, UK) was used for transfection and greenfluorescence of transfectant pool was measured in a FACS with excitationat 488 nm. For every GS/GFP vector construct, transfection was carriedout independently five times, all data being the average from fiveindependently analyzed pools. Starting with transient transfectants 48 hpost-transfection, the top scoring 10% highly expressing cells of theviable cell pool in the cell count vs. fluorescence diagram wereselected to determine mean fluorescence (FIG. 1). Viable cell populationhas been preselected by gating in the Forward vs. sideward scatterdiagram.

For generating stable transfectants, GS marker was selected 24 hourspost-transfection by supplementing the glutamine-free medium with 25 μMMSX (methionine sulphoximine, Crockett et al., ibd.) and continuing cellculture with regular splitting of cultures for 26 days. Note the impactof medium levels of other amino acids on the potency of MSX forselection, see Bebbington et al., U.S. Pat. No. 5,827,739. Flurorescenceanalysis was then performed again as outlined above (FIG. 2).

Untransfected cells served as negative control. The hot spot vector (pEE15.1 ‘hCMV+hot spot’) driving expression of GFP under control of thehCMV promoter comprising the first complete intron of CMV is given inSeq. ID No. 1 and essentially is the pEE 15.1 vector shown in FIG. 3into which the GFP sequence was inserted into the Eco RI restrictionsite in the polylinker. pEE 12.4 ‘hCMV’ corresponding to Seq. ID No. 3is identical to pEE 15.1 ‘hCMV+hot spot’ except that it does notcomprise the 5.1 kb Bam H1 fragment harboring the IgA 2A sequence. pEE12.4 served as a vector control. A further vector control pEE 12.4‘hCMV(Kozak-)’ was generated by mutating the Kozak sequence of thecloning site coninciding with the translation start site (GCCGCCACCATGG)to a frameshifted functional Kozak sequence that (ACCATGGGTCCATGG) byprimer directed mutagensis (Sambrook et al., Molecular cloning, ColdSpring Harbor 1983), attentuating the original Kozak and translationstart site. The vector of Seq. ID No. 1 was further engineered to deletethe 400 bp modulator region of hCMV enhancer portion, deleting theenhancer elements upstream of −750 from the transcription start site,giving rise to pEE 15.1 ‘hCMV(mod-)/GS cDNA’. By exchange of the GScDNAcassette of pEE 15.1 (s. FIG. 3) with the GS minigene of pEE 14.4‘hCMV(mod-)’/GFP, corresponding to Seq. ID No. 2, the vector pEE 15.1‘hCMV(mod-)/GS minigene’ was created. Thus all transfected cellsharbored a plasmid vector comprising the GFP coding sequence. The GSminigene contains a single, first intron of the GS gene and about 1 kbof 3′ flanking DNA under the control of the SV40 late promoter; the 3′part of the genomic GS DNA is believed to cause a higher copy-number ofvector DNA and thus of GS in transfected cells (see U.S. Pat. No.4,770,359, Bebbington et al.). Whereas all hCMV vectors employed in thepresent study express the GS maker gene from its cDNA sequence, use ofthe GS minigene was included as a further control in order to excludepotential effects of GS copy number and expression level.

For generation and expression analysis of stably transfected CHO cells,transfections were performed with linearized hot spot vector pEE 15.1‘hCMV+hot spot’ vector. Sal I linearized plasmid was cut in the IgA 2Acomprising sequence portion, free DNA ends potentially stimulatingrecombination with genomic regions sharing a certain degree of homologywith the flanking DNA portions, testing for potential targetting effectsof murine IgG 2A in hamster CHO cells. Pvu I cut in the bacteriallactamase marker gene and therefore could promote but heterologousrandom recombination. Indeed, the mean fluorescence was higher in thePvu I linearized transfectants showing both some influence of vectorlinearization as well as that targetting to immunoglobuline loci in CHOcells may not account for the effect of the present invention. Inaddition, the effect of enhanced promoter activity was consistinglyobserved in transiently transfected cell populations, nicely correlatingwith relative strength of individual vector constructs. Clearly, genomicintegration is not involved at this early stage of transfection.

FIG. 3 shows vector pEE 15.1 of approximately 12 830 bp. A detaileddescription of the GS marker and the hCMV-p/intron expression cassettecan be found in U.S. Pat. No. 5,827,739 and U.S. Pat. No. 5,591,639. pEE15.1.is a possible embodiment of an expression vector according to thepresent invention, except that the DNA sequence coding for therecombinant product protein has not yet been inserted in the polylinkersite. The complete 13535 bp sequence of the pEE 15.1 construct harboringGFP is given in Seq. ID No. 1: Therein, the GFP coding sequence wasinserted in-frame in the Eco R I restriction site centered at baseposition 12 814; the introduction of the unique restriction siteharboring the ATG start codon and optimizing the Kozak sequenceenvironment of the start codon is described in detail in U.S. Pat. No.5,591,639. Thus, the expression of GFP protein is under control of thehCMV-major immediate early gene promoter (HCMV-MIE or hCMV for short)immediately followed by the first intron of hCMV-MIE gene followed bythe Nco I site (s. U.S. Pat. No. 5,591,639).

Polyadenlyation is ensured by the SV40 poly A site further downstream ofthe polylinker insertion site. pEE 15.1 further harbors a cDNA sequencecoding for glutamine synthetase (GS) from hamster that is under controlof the SV40 early promoter and is followed by an SV40 intron+poly Asequence. The IgG 2A gene locus or ‘hot spot’ sequence (hatched boxesCH1, Hi, CH2, CH3, M1, M2 standing for Heavy chain constant region,hinge, membrane anchor) is the 5.1 kb BamH I fragment of the murine IgG2A locus already described in WO 9517516 and the references citedtherein. Unique restriction sites Pvu I and Sal I are shown.

Experiment 2

Electroporation of CHO Cells With mCMV p12.4 -GFP Construct (Seq. IDNo.4)

Attached CHO-K1 cells (ATCC CCL-61) were cultured in Iscoves' DMEMmedium essentially as described in EP-481 791 comprising 2 mM Glutaminewhich was further supplemented with 10% FCS. Optionally, the G-MEMmedium stated in table 1 and further comprising 2 mM Glutamine could beused prior to GS marker selection as in experiment 1. The cells weredetached, pelleted and resuspended twice in serum-free medium, finallyat a density of 5.3×10⁶ cells/ml. Per 750 μl electroporation batch, atotal of 4×10⁶ cells was electroporated. Electroporation was carried outas described in Methods in Molecular Biology, ed. JA Nickoloff ed,Humana Press 1995, Vol. 48/Chap. 8: Animal cell electroporation andelectrofusion protocols. p12.4 mCMV-GFP vector DNA (sequence ID No. 4)was linearized. 50 μl (20 μg) DNA were added to 750 μl cells inelectroporation cuvette and electroporate—300 Volts/750 μFd—expecting anelectorporation time of around 12-14 msec. Following electroporation 800μl volume of cells was transferred into 25 ml of modified Glasgow-MEM(GMEM, Gibco) culture medium for GS selection (comprising 10% fetalserum but no glutamine, for details see table 1 ) in a T75 flask. Divideinto 2×T75 flasks by moving 12.9 mls into a second flask and incubateovernight at 37° C. in 10% CO₂

On the next day 37.5 ml of GS-selection GMEM culture medium supplementedwith 10% FBS+33.3 μM MSX (methionine sulphoximine) were added. Thus MSXwas finally ˜25 μM. Transfectants were counted after further incubationfor 26 days by colony count per flask. Upon microscopic inspection in astandard inverted microscope for inspection of culture flasks, positivecolonies brightly lit up in light green and could be easily counted.

The mCMV construct of Seq. ID No. 4 yielded up to 20 times more focithan did cells that were transfected in parallel with the hCMV constructof Seq. ID No. 3. The vector constructs only differed in the CMVpromoter elements driving GFP expression, the remaining vector parts ofthe vectors were identical (including GS-marker; cDNA GS-marker cassetteof p12.4). If cells were diluted out into 96 well plates immediatelyfollowing transfection, many more colonies come up from mCMV transfectedcells (>400 colonies) than from hCMV transfected cells (about 45colonies). TABLE 1 Medium for GS selection A. Stock Solutions 1. Doubledistilled water autoclaved in 400 ml aliquots 2. 10 × Glasgow MEM (GMEM)without glutamine (GIBCO: 042-2541 in UK). Store at 4° C. 3. 7.5% sodiumbicarbonate (GIBCO: 043-05080 in UK; 670-5080 in US). Store at 4° C. 4.100 × non-essential amino acids (NEAA) (GIBCO: 043-01140 in UK; 320-1140in US). Store at 4° C. 5. 100 × Glutamate + Asparagine (G + A): add 600mg glutamic acid and 600 mg asparagines (Sigma). Make up to 100 ml indistilled water and sterilize by passing through a sterile 2 μm filter(Nalgene). Store at 4° C. 6. 100 mM sodium pyruvate (GIBCO: 043-01360 inUK; 320-1360 in US) 7. 50 × nucleosides: 35 mg adenosine 35 mg guanosine35 mg cytidine 35 mg uridine 12 mg thymidine (each from Sigma). Make upto 100 ml with water, filter sterilise and store at −20° C. in 10 mlaliquots. 8. Dialysed PCS (GIBCO: 014-06300). Heat inactivate at 56° C.for 30 min and store at −20° C. It is essential to use dialysed FCS whenusing GS selection. 9. Penicilin-streptomycin at 5000 units/ml (P/S:GIBCO: 043-05070 in UK; 600-5070 in US). 10. 100 mM L.MSX (Sigma):prepare 18 mg/ml solution in PBS. Filter sterilise and store at −20° C.B.Medium Preparation

Add the following in the order given using aseptic technique to makeGMEM-S medium 1. Water 400 ml 2. 10 × GMEM 50 ml 3. Sodium bicarbonate18.1 ml 4. NEAA 5 ml 5. G + A 5 ml 6. Sodium pyruvate 5 ml 7.Nucleosides 10 ml 8. Dialysed FCS 50 ml 9. Penicillin-streptomycin 5 mlGMEM-S contains the non-essential amino acids, alanine, aspartate,glycine, proline and serine (100 μM), glutamate and asparagines (500μM), and adenosine, guanosine, cytidine and uridine (30 μM), andthymidine (10 μM).

1-8. (canceled)
 9. CHO cell transfected with an expression vectorcomprising a promoter that is active in CHO cells and that is drivingexpression of a recombinant product protein and further comprising aportion from the murine IgA 2A gene locus DNA which portion is enhancingactivity of said promoter.
 10. CHO cell according to claim 9,characterized in that the vector further comprises a transcription unitencoding a selectable marker, preferably a glutamin synthetase (GS)marker.
 11. CHO cell according to claim 9 or 10, characterized in theCHO cell is stably transfected.
 12. Method of expressing a recombinantprotein, comprising the steps of culturing a CHO cell transfected withan expression vector comprising a promoter active in CHO cells drivingexpression of a recombinant product protein and further comprising themurine IgA 2A gene locus DNA or a DNA sequence variant or DNA fragmentthereof which is enhancing activity of said promoter, and harvesting theproduct protein
 13. Method according to claim 12, characterised in thatthe promoter is a strong viral promoter, preferably the hCMV promoter.14. Method according to one of claims 12 or 13, characterised in thatthe IgA 2A gene locus portion does lack the natural immunoglobulinpromoter.
 15. Method according to claim 12, characterized in that thepromoter is hCMV promoter or a functional part thereof having promoteractivity wherein said promoter or functional part lack the ‘modulator’sequence in the upstream/enhancer portion as found stretching fromposition −750 to −1150 relative to the MIE transcription start site. 16.CHO cell transfected with a mammalian expression vector comprising atleast a first transcription unit for a product gene which transcriptionunit is under the control of the mCMV promoter, and further comprising asecond transcription unit comprising a glutamine synthetase (GS) markergene.
 17. Mammalian expression vector comprising at least a firsttranscription unit for a product gene which transcription unit is undercontrol of the mCMV promoter or a functional fragment thereof, andfurther comprising a second transcription unit comprising a glutaminesynthetase (GS) marker gene.
 18. Vector according to claim 16, whereinthe mCMV promoter or functional fragment comprises the naturaltranscription start site (+0) and extends upstream to position −500. 19.Vector according to claim 18, wherein the mCMV promoter or functionalfragment extends to the natural Xho l restriction site.
 20. Vectoraccording to claim 18, wherein the transcription start site isengineered to comprise a suitable restriction site for insertion of arecombinant gene product.
 21. Vector according to claim 17 or 18,wherein the first transcription unit harbors at least one intronsequence.
 22. Vector according to claim 21, wherein said intron is notthe first, natural intron of the mCMV promoter.
 23. Method of using 17for enhancing the transfection rate in CHO cells.